DNA-binding specificity and molecular functions of NAC transcription factors

The family of NAC (NAM/ATAF1,2/CUC2) transcription factors has been implicated in a wide range of plant processes, but knowledge on the DNA-binding properties of the family is limited. Using a reiterative selection procedure on random oligonucleotides, we have identified consensus binding sites for two NAC proteins. The consensus sequences are similar, but not identical; both contain the core CGT[GA]. The strict consensus sequences, comprising only the most frequent base at each position, are: TTNCGTA and TTGCGTGT. In silico analysis of target promoter regions corroborated the selection results. Furthermore, NAC protein binding to the CaMV 35S promoter was shown to depend on sequences similar to the consensus of the selected oligonucleotides. Electrophoretic mobility shift assays demonstrated that NAC proteins bind DNA as homo- or heterodimers and that dimerization is necessary for stable DNA binding. The ability of NAC proteins to dimerize and to bind DNA was analysed by structure-based mutagenesis. This identified two salt bridge-forming residues essential for NAC protein dimerization. Alteration of basic residues in a loop region containing several highly conserved residues abolished DNA binding. Thus, the results presented here contribute significantly to our understanding of the specificity and molecular functions of the NAC protein DNA-binding domain.